The success of kinases as drug targets continues to motivate the development of new techniques to discover kinase inhibitors (1-3). Biochemical assays that show excellent sensitivity and are non-hazardous and low cost are needed (4). In addition, the different ways that inhibitors can bind to kinases (ATP-competitive, allosteric, inactive conformation, etc.) makes employing multiple approaches to characterize inhibitors advantageous (5, 6). For instance, in order to characterize inactive state inhibitors, methods that directly measure binding, rather than inhibition of activity, are desirable (1, 7, 8).
Several methods that have previously been employed to study direct binding of kinase inhibitors include isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and fluorescence competition (9-12). Each of these approaches has advantages as well as disadvantages. ITC can be performed in solution but is low through-put and requires a significant amount of material. SPR is higher-throughput but requires attachment of the kinase to a surface. A promising new method is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR-FRET) (13, 14). In this method, a FRET donor such as europium cryptate is bound to the kinase via interaction with a purification tag (FIG. 1A). A FRET acceptor such as Alexa Fluor 647 is also bound to the kinase via attachment to a known inhibitor and serves as a tracer. If the 2 fluorophores are in proximity with the proper orientation, FRET will occur between them (FIG. 1A). Displacement of the tracer with an unlabeled inhibitor eliminates the FRET signal and provides a read-out on binding. This FRET-based competition assay has many potential advantages over competing methods; it is homogeneous, non-hazardous, high-throughput, and potentially has high sensitivity allowing for low usage of material and low cost.
FRET-based competition has previously been utilized in a few cases to characterize interaction of inhibitors with kinases. For instance, investigators have coupled europium via an AviTag-mediated, biotin-streptavidin interaction to the N-terminus of p38α kinase and proceeded to monitor inhibitor binding by competitive displacement of an Alexa Fluor 647-coupled p38α inhibitor (13). In another study, europium was coupled to kinases via an N-terminal GST/Anti-GST antibody interaction and inhibitor binding to a panel of kinases was studied by monitoring displacement of Alexa Fluor 647-tagged staurosporine (14). These studies have established the feasibility of using FRET-based competitive binding to characterize kinase inhibitors. However, there are many examples whereby binding both a tracer and europium does not result in a sufficient FRET signal. Hence, it would be useful to identify the most promising tagging strategies that reliably result in a robust FRET signal.